Apolipoprotein (apo) E serves as a ligand to mediate the clearance of triglyceride-rich lipoproteins, such as very low density lipoproteins (VLDL) and their remnants, from circulation. Paradoxically, however, we have recently found that overexpression of apoE3 causes hypertriglyceridemia in transgenic mice or combined hyperlipidemia in transgenic rabbits by directly stimulating hepatic VLDL production and by impairing VLDL lipolysis. The goal of this proposal is to investigate the molecular mechanisms by which apoE expression levels determine VLDL metabolism. The first aim is to establish quantitatively the effects of increasing apoE expression levels on VLDL production, lipolysis, and clearance in mice. We will test the hypothesis that all three effects of apoE on VLDL metabolism are dependent on apoE expression levels. We will determine the range of apoE levels that support normal VLDL and remnant lipoprotein clearance and that inhibit lipolytic processing by increasing plasma apoE levels from extrahepatic sources. We will also determine the effect of increasing hepatic apoE synthesis on the induction of VLDL production. The second aim is to investigate the cellular and molecular mechanisms responsible for the stimulation of hepatic VLDL production by apoE. Hepatic VLDL production appears to involve two steps and different regulatory factors. We will determine the effects of apoE on each step and on several regulatory factors in transfected rat hepatoma (McA-RH7777) cells expressing different levels of human apoE. We will address the following three major questions: Does apoE stimulate triglyceride synthesis in the cells? Does apoE transport the newly synthesized triglycerides within the cells? Does apoE colocalize with apoB in the McA-RH7777 cells? Recently, three new polymorphisms (-491A/T, -427T/C, and -219T/G) have been found in the transcriptional regulatory region of the apoE gene. The third aim is to test the hypothesis that the apoE promoter polymorphisms may affect plasma levels of apoE and VLDL metabolism in humans. We will determine the allelic frequency of apoE promoter polymorphisms in normolipidemic humans by using restriction fragment length polymorphism analysis and the effect of apoE promoter polymorphisms on plasma levels of apoE and lipoproteins in normolipidemic and hyperlipidemic subjects.